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Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
Int. J. Cancer (Pred. Oncol.): 74, 45–49 (1997)
r 1997 Wiley-Liss, Inc.
DELETION OF THREE DISTINCT REGIONS ON CHROMOSOME 13q
IN HUMAN NON-SMALL-CELL LUNG CANCER
Kenji TAMURA,1 Xue ZHANG,1 Yoshinori MURAKAMI,1 Setsuo HIROHASHI,2 Hong-Ji XU,3
Shi-Xue HU4, William F. BENEDICT4 and Takao SEKIYA1*
1Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan
2Pathology Division, National Cancer Center Research Institute, Tokyo, Japan
3M.D. Anderson Cancer Center, The Woodlands, TX, USA
We examined loss of heterozygosity (LOH) at the retinoblastoma susceptibility gene (RB1) locus on chromosome
13q14 in 20 non-small-cell lung cancers (NSCLCs) using
polymorphic markers. The expression of RB protein was
examined by immunohistochemical analysis of paraffinembedded specimens of the same tumors. The results revealed that 10 of 16 informative cases showed an LOH at the
RB1 locus, whereas only 2 of the 10 tumors lost expression of
the RB protein. These 2 tumors had mutations in the
remaining RB1 allele. Thus, inactivation of the RB1 gene
appears to be involved in a small subset of NSCLCs only. To
elucidate the presence of tumor-suppressor genes other than
RB1 on 13q, heterozygosity at 15 different loci was investigated. Of 20 tumors analyzed, 15 showed an LOH at least at
one locus, and the regions 13q12.1-qter, 13q12.2-14.2 and
13q14.1-q14.3, including the RB1 locus, were deleted in
significant numbers of the tumors. Our results suggest that,
in addition to the RB1 gene, abnormalities of other tumorsuppressor genes on chromosome 13q are involved in the
development of human NSCLCs. Int. J. Cancer 74:45–49.
r 1997 Wiley-Liss, Inc.
The retinoblastoma susceptibility gene (RB1), which is located
on the long arm of chromosome 13 (q14), is the first tumorsuppressor gene identified in human cancers (Friend et al., 1986;
Fung et al., 1987; Lee et al., 1987). RB1 gene inactivation was
revealed not only in retinoblastoma but also in many other human
tumors, including osteosarcomas; soft tissue sarcomas; small-cell
lung carcinomas; and breast, bladder, prostate, liver and esophageal
cancers (see review: Benedict et al., 1990).
In non-small-cell lung cancers (NSCLCs), Xu et al. (1991) and
Reissman et al. (1993) have shown the absence of RB protein by
immunohistochemical means in about one-third of tumors. We
previously identified an LOH at the RB1 locus in 6 of 8 informative
squamous-cell lung cancers. However, PCR-SSCP analysis of the
remaining RB1 gene revealed inactivating mutations in only 2 of
the 6 tumors (Sachse et al., 1994). The discrepancy between LOH
at the RB1 locus and the loss of RB protein expression has also
been reported on larger series of NSCLCs. Reissmann et al. (1993)
demonstrated that only 8 of 25 tumors with loss of expression of the
RB protein showed LOH at the RB1 locus, while one of 12 tumors
with normal RB protein expression showed LOH at the RB1 locus.
Gouyer et al. (1994) also demonstrated LOH at the RB1 locus in 11
NSCLC tumors, only 4 of which showed absence of the RB
protein.
In this study, we examined the allelic states of different loci on
chromosome 13q together with the RB1 locus and the RB protein
status in human NSCLCs. Our results indicated that inactivation of
the RB1 gene was involved in only a subset of NSCLCs and that the
presence of commonly deleted regions carried putative tumorsuppressor genes other than RB1.
MATERIAL AND METHODS
DNA samples
Cancerous and non-cancerous tissues of the lung were resected
surgically or obtained at autopsy from 20 patients with NSCLC at
the National Cancer Hospital (Tokyo, Japan). High m.w. DNA was
prepared from these specimens by the proteinase K-phenolchloroform extraction method. These cancers included 9 adenocarcinomas, 6 squamous-cell carcinomas, 2 adenosquamous carcinomas, 1 adenoid cystic carcinoma and 2 large-cell carcinomas.
LOH analysis with polymorphic markers
Nineteen polymorphic markers, 4 at the RB1 locus and 15 at
other loci on chromosome 13q, were amplified by PCR for LOH
analysis. Sixteen markers detected polymorphisms in microsatellite
repeating sequences, while 2 and 1 detected those of single-base
substitutions and variable (T)s, respectively. These features are
summarized in Table I. Primers with reported nucleotide sequences
(Murakami et al., 1991; Sachse et al., 1994; Zhang et al., 1994)
were supplied by Bio-Synthesis (Lewisville, TX) and used to
amplify DNA markers. The PCR mixture of 5 µl contained 50 ng of
template DNA and primers labeled by the polynucleotide kinase
reaction with [g-32P]ATP, as described (Orita et al., 1989b). Thirty
cycles of the reaction proceeded at 94°C for 20 sec and at 56–60°C
for 2 min in a Gene Amp PCR system 9600 (Perkin-Elmer Cetus,
Emeryville, CA). The reaction mixture was diluted 10-fold with
95% formamide, 20 mM EDTA, 0.05% xylene cyanol and 0.05%
bromophenol blue, then heated at 80–90°C for 5 min. To analyze
DNA markers with microsatellite sequences and variable Ts, 1 µl of
the diluted mixture was loaded onto a 5% polyacrylamide gel
containing 7 M urea and resolved by electrophoresis at 50°C for
70–130 min. To detect a single-base substitution polymorphism by
SSCP analysis (Orita et al., 1989a,b), the diluted mixture (1 µl) was
subjected to electrophoresis in a non-denaturing 5% polyacrylamide gel containing 5% glycerol at 10°C for 200 min. These gels
were dried on filter paper and exposed to Kodak XAR-5 film
(Eastman-Kodak, Rochester, NY) at room temperature for 5–8 hr.
Immunohistochemical staining
RB nuclear protein was stained in paraffin-embedded sections as
described (Xu et al., 1991), using the highly specific, affinitypurified, polyclonal anti-RB antibody RB-WL-1 (Zhang et al.,
1994).
Contract grant sponsor: Ministry of Health and Welfare of Japan
grant-in-aid for the 2nd Comprehensive 10-Year Strategy for Cancer
Control; Contract grant sponsor: Ministry of Health and Welfare of Japan
grant for research on aging and Health; Contract grant sponsor: NIH
National Cancer Institute, contract grant number CA54672.
Xue Zhang’s present address is Department of Medical Genetics, China
Medical University, Shenyang 110001, People’s Republic of China.
*Correspondence to: Takao Sekiya, Oncogene Division, National Cancer
Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104,
Japan. Fax: 81-3-5565-9535.
Received 28 June 1996; Revised 2 October 1996
TAMURA ET AL.
46
TABLE I – POLYMORPHIC MARKERS ON THE LONG ARM OF CHROMOSOME 13
3
3
Locus symbol
Location
Polymorphism
Size range (bp)
D13S141 4
D13S120
D13S139
D13S127
D13S126
D13S118
RB 1 (RB 1.20)
RB 1 (RB intron 11)
RB 1 (RB intron 21)
RB 1 (RB intron 25)
D13S227
D13S228 4
D13S133
D13S137
D13S119
D13S146
D13S131
D13S121
13q11
13q11–12.1
13q12.1
13q12.3–14.3
13q12.2–14.1
13q14.1–14.2
13q14.1–14.2
13q14.3
13q14.3
13q14.3
13q14.3
13q14.3
13q14.3
13q14.3
13q14.3
13q14.3–22
13q21.1–22
13q21.1–22
13q31
(CA)n
(CA)n
(CA)n
(CA)n
(CA)n
(CA)n
(CA)n
(CTTT)n
C/T
Tn
T/A
(CA)n
(CA)n
(CA)n
(CA)n
(CA)n
(CA)n
(CA)n
(CA)n
100–109
125–127
112–136
129–137
130–142
100–112
187–201
550–600
245
417
202
76–85
130–134
134–185
117–127
124–140
103–134
166–172
160–178
D13S175 1
1Brackets
indicate that the order of the loci has not been determined.
RESULTS
Status of the RB1 gene and RB protein in NSCLCs
The allelic state of RB1 was analyzed in 20 human NSCLCs
using 4 polymorphic markers within the gene. As a representative
result, detection of microsatellite sequence polymorphism and
LOH by RB1.20 is shown in Figure 1. Similar analyses revealed
LOH in the RB1 gene in 10 of 16 informative cases, as summarized
in Table II.
To establish whether the tumors with LOH at the RB1 locus also
had mutations in the remaining RB1 allele, expression of the RB
protein in tumors was examined immunohistochemically using
paraffin-embedded specimens of the same set of tumors and
polyclonal antibody against RB protein RB-WL-1. Representative
results are shown in Figure 2. The nuclei of the cancer cells without
loss of the RB1 allele as well as those of normal parenchymal cells
of patient 141 were heterogeneously stained with the antibody (Fig.
2a). However, cancer cells with an allelic loss of the RB1 gene from
patient 167 were not stained with the antibody, indicating inactivation of the remaining RB gene (Fig. 2b). In the patient, noncancerous cells surrounding the cancer cells were clearly stained.
The results of the immunohistochemical analysis are summarized
in Table II. All of the tumors that retained heterozygosity at the RB1
locus expressed RB protein. However, of 10 tumors with an LOH at
the RB1 locus, only 2 that were known to have the mutated RB1
gene did not express RB protein.
Analysis of LOH on chromosome 13q
DNA from the same 20 patients with NSCLCs was examined for
an LOH on 13q at various loci using 15 markers for polymorphic
sequences together with polymorphic markers within the RB1 gene.
Representative results of the LOH analysis are shown in Figure 3.
A comparison of cancerous and normal DNA from patient 206
revealed an LOH at the D13S175 locus in DNA from tumors, while
the DNA retained heterozygosity at the D13S120 locus. In tumor
LuC137, there were LOHs at the RB1 and D13S228 loci but
heterozygosity at the D13S133 locus was retained. Tumor LuC169
showed LOH at the D13S139 locus but not at either the D13S120 or
the RB1 locus.
Our results are summarized in Figure 4. Fifteen of 20 tumors had
an LOH at least at one 13q locus analyzed. Five of them showed an
LOH at all of the loci examined, suggesting loss of the entire
chromosome 13. The results of a partial loss of chromosome 13q in
the other 10 tumors indicated the presence of 3 separate regions
commonly deleted in significant numbers of tumors. Regions I, II
and III were from the centromeric region of 13q to the D13S120
locus (13q12.1), between the D13S139 (13q12.3–14.3) and D13S118
FIGURE 1 – Allelic losses at the RB1 locus in human NSCLCs.
Polymorphic RB1.20 sequence was amplified by PCR from pairs of
DNA from cancerous and non-cancerous tissues of patients. Heterozygosity at the RB1 locus was retained in the tumor in patient 169, while
LOH was observed in the tumor in patient 137. N and C represent DNA
from non-cancerous and cancerous tissues, respectively.
TABLE II – ALLELLIC LOSS AT THE RB LOCUS, LOSS OF RB PROTEIN
Tumor
Histological type1
Stage
Allelic loss2
RB
protein
Inactivating
mutation5
LuC59
LuC104
LuC118
LuC128
LuC137
LuC141
LuC148
LuC150
LuC153
LuC161
LuC164
LuC166
LuC167
LuC168
LuC169
LuC190
LuC195
LuC200
LuC202
LuC206
Ad
Adenosq
La
La
Adenoid cyst
Sq
Sq
Sq
Ad
Sq
Ad
Adenosq
Sq
Ad
Ad
Sq
Ad
Ad
Ad
Ad
IV
IV
I
I
IIIa
IIIa
I
IIIa
IV
I
IIIa
II
I
I
I
IIIa
I
IV
IIIa
IIIa
1
NI3
2
ND
1
1
1
1
NI
1
2
1
1
1
2
1
2
2
2
2
ND4
1
1
1
1
1
1
2
1
1
1
1
2
ND
1
1
1
1
1
1
2
26
2
2
2
2
2
17
2
2
2
2
18
2
2
2
2
2
26
2
1Ad, adenocarcinoma; Adenosq, adenosquamous-cell carcinoma;
La, large-cell carcinoma; Adenoid cyst, adenoid cystic carcinoma; Sq,
squamous-cell carcinoma.—21 and 2 indicate allelic loss and retention of 2 alleles, respectively.—3NI, not informative.–4ND, not done.–
5Mutations were detected previously (Sachse et al., 1994).—6LuC104
and LuC202 carried cancer-specific mutations with unknown function,
CAC to CCC (codon 673, exon 20) and G to T (intron 14),
respectively.–7Abnormal splicing; GTAAG to GCAAG (intron 17).—
8Frameshift: AAG to AG (codon 96, exon 3).
DELETION OF 13q IN HUMAN NSCLC
47
FIGURE 2 – Immunohistochemical staining of RB nuclear protein. (above) An RB-positive tumor, LuC 141, showed an LOH at the RB1 locus
but no mutations in the remaining allele. (below) An RB-negative tumor, LuC 167, showed an LOH at the RB1 locus and a mutation in the
remaining allele. RB protein expression was detected by staining with the antibody RB-WL-1. Scale bar: 10 µm.
(13q14.1–14.2) loci and between the D13S118 (13q14.1–14.2) and
D13S133 (13q14.3) loci, respectively. Region III includes the RB1
locus.
DISCUSSION
Tumor tissues in which DNAs were analyzed for LOH might
have contained normal cells. In contrast, immunohistochemical
analysis of paraffin-embedded sections of these specimens was
straightforward and would detect expression of the RB protein
directly in each of the cancer cells. Therefore, the results obtained
for RB protein were unambiguous. Thus, immunohistochemical
analysis has been used as a sensitive method for detection of RB1
gene inactivation, though the results of the analyses are known to
be dependent on the quality of the antibodies tested (Xu et al.,
1991; Reissmann et al., 1993; Geradts et al., 1996). In the present
study, we examined expression of the RB protein with anti-RB
antibody, RB-WL-1, and confirmed the discrepancy between
LOH at the RB1 locus and loss of expression of the RB protein.
This antibody has been widely used to detect RB protein expression in various tumors (Xu et al., 1991, 1993, 1994; Zhang et al.,
1994).
In this study, we detected 2 tumors, LuC150 and LuC167,
lacking RB protein expression. Only these tumors showed an LOH
48
TAMURA ET AL.
FIGURE 3 – Allelic losses at loci on chromosome 13q in human
NSCLCs. Polymorphic sequences were amplified by PCR from pairs of
DNA samples from cancerous and non-cancerous tissues of patients.
Products were resolved by denaturing polyacrylamide gel electrophoresis. N and C represent DNA from non-cancerous and cancerous
sources, respectively.
at the RB1 locus as well as an inactivating mutation in the
remaining RB1 allele (Table II). The coincident results indicate that
antibody RB-WL-1 is highly specific to RB protein. In contrast,
RB protein was detected in the other tumors with an LOH at the
RB1 locus. These results indicated that inactivation of the RB1
gene was certainly involved but in only a subset of NSCLCs.
We identified 3 commonly deleted regions (I–III) by extensive
analyses of LOH on chromosome 13q. LOHs could be produced by
either interstitial deletion or translocation of the chromosome. It
may be possible that chromosome 13 contains unstable regions
where chromosomal breakage could occur with a high frequency.
As shown in Figure 4, region I was suggested by LOHs in tumors
LuC150, LuC202 and LuC206; region II by those in LuC164,
LuC169 and LuC202; and region III by those in LuC137, LuC153
and LuC166. Regions I and II, which might carry tumor-suppressor
genes other than RB1, were located closer to the centromere, while
region III included the RB1 locus. However, in the 3 tumors
carrying LOH at region III, RB protein was expressed. Therefore,
the presence of a tumor-suppressor gene other than RB1 was
suggested.
Frequent LOH on chromosome 13q and less frequent RB1
mutations have also been found in various human tumors, including breast cancers (Borg et al., 1992), head and neck cancers (Yoo
et al., 1994) and pituitary tumors (Pei et al., 1995). Detailed
FIGURE 4 – Allelic losses at the loci on the long arm of chromosome
13 in NSCLCs. Patient numbers are indicated at the top of the panel.
Loci analyzed are indicated at the left side of the panel. Loci in the box
have not yet been placed in order. d and s, Loss and retention of
heterozygosity, respectively. Vertical lines indicate loci where the
signals were not informative. Solid bars on the right indicate commonly deleted regions.
analyses of head and neck cancers and pituitary tumors have
indicated that commonly deleted regions also include the RB1
locus and overlap region III identified in the present study. Schutte
et al. (1995) have reported that a restricted fragment within region
II at 13q12.3 is homozygously deleted in human pancreatic
cancers. In this region, the BRCA2 gene for susceptibility to
hereditary breast cancer has been identified (Wooster et al., 1995).
It has been reported that the BRCA2 gene and the RB1 gene could
be distinct target loci for allelic imbalance at chromosome 13 in
sporadic breast cancer (Hammann et al., 1996). The relationship
between tumor-suppressor genes on 13q in NSCLCs and those in
other tumors remains to be clarified.
ACKNOWLEDGEMENTS
This work was supported in part by a grant-in-aid for the 2nd
Comprehensive 10-Year Strategy for Cancer Control and a Grant
for Research on Aging and Health, both from the Ministry of
Health and Welfare of Japan, and grant CA54672 from the National
Cancer Institute, NIH (to W.F.B.). K.T. was the recipient of a
Research Resident Fellowship from the Foundation for Promotion
of Cancer Research, Japan.
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